Abstract The lipid composition of the membrane bilayer surrounding different cellular organelles is unique both in terms of structural lipids and signaling lipids like the phosphoinositides, lending each compartment distinct biochemical and biophysical characteristics intrinsic to its function. In this MIRA proposal, we address the fundamental and largely unexplored question of how cells maintain these distinct lipid compositions, even in light of continuous vesicle trafficking and lipid exchange between compartments. Our research will focus on two poorly understood mechanisms for controlling lipid homeostasis: lipid exchange at membrane contact sites and lipid remodeling by multi-functional phosphoinositide kinase/phosphatase complexes. Membrane contact sites, where two organelles come into close apposition, are emerging to play a critical role in membrane lipid dynamics and homeostasis. To discover the processes occurring at such sites and their molecular basis, we are exploring which proteins localize there, what their function is, how and when are they recruited there, and how their activity is regulated. Our studies in the next project period will focus on VPS13 and related proteins, suggested by our preliminary data to comprise a new family of lipid transport proteins. These studies promise exciting new insights into membrane biology, including for the long-standing questions of how mitochondria and the autophagosomal isolation membrane, neither connected to well-established vesicular trafficking pathways, may acquire their membrane lipids. Membrane contact sites can also modulate the levels of phosphoinositide lipid species present at different compartments, but regulation by lipid kinases and lipid phosphatases peripherally associated with the membrane bilayer of individual organelles likely plays a more significant role in controlling local phosphoinositide levels. To better understand the mechanisms governing phosphoinositide homeostasis, we are characterizing these enzymes, which reversibly interconvert phosphoinositide species via the phosphorylation and dephosphorylation of their inositol headgroups. In particular, in the next project period, we will focus on how levels of phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2), which plays a central role in the biology of the lysosome/vacuole, are regulated by the PIKfyve complex. The mechanisms underlying PI(3,5)P2 metabolism have been elusive, owing in part to the complexity of this assembly which comprises at least three different proteins and antagonistic lipid kinase and lipid phosphatase activities. Studying this complex in vitro, separate from the many processes ongoing in living cells, will be critical in understanding how PI(3,5)P2 synthesis and degradation are individually regulated and ultimately coordinated. For these projects, we will leverage our expertise in structural, biochemical, and biophysical techniques in vitro, then test arising hypotheses functionally via well-established collaborations or in consultation with cell biologist colleagues.